Renal nuclear matrix proteins, polynucleotide sequences encoding them, and their use

ABSTRACT

Nuclear matrix proteins (NMP) are useful markers in diagnosing and monitoring the stage of malignancy of a cell, and in treating cell proliferative disorders associated with the NMP.

[0001] This invention was made with support from the National KidneyFund of Western Pennsylvania and from the National Kidney CancerAssociation.

BACKGROUND OF THE INVENTION

[0002] The present invention relates generally to renal nuclear matrixproteins, called “NMPs” here, and more specifically to novel nuclearmatrix proteins of the kidney which are associated withcell-proliferative disorders.

[0003] U.S. Pat. Nos. 4,882,268 and 4,885,236, both issued to Fey andPenman, disclose certain NMPs and discuss their possible uses inidentifying the origin of a tissue sample. In addition to identifyingthe origin of a tissue sample, the patents also discuss possible uses ofNMPs to indicate certain disease states of a cell, such as viralinfection, cancer, chromosomal defects or autoimmune disease. SpecificNMPs discussed in the patents include those from human colon, lung,adrenal cortex and bladder cell lines.

[0004] The early diagnosis of renal cancer is central to the effectivetreatment of the disease. Currently, there are no methods available toeasily and specifically identify the presence of renal cancer cellsbased on NMPs.

[0005] The nuclear matrix is the structural component of the nucleusthat determines nuclear morphology, organizes the DNA in athree-dimensional fashion that is tissue specific, and has a centralrole in the regulation of a number of nuclear processes including theregulation of gene expression. The nuclear matrix has been demonstratedto play a central role in the regulation of important cellular processessuch as DNA replication and transcription. Getzenberg, J. Cell Biochem.55: 22-31 (1994). The nuclear matrix is the framework or scaffolding ofthe nucleus and consists of the peripheral laminas and pore complexes,an internal ribonucleic protein network, and residual nucleoli. Berezneyet al., Biochem. Biophys. Res. Comm. 60: 1410-17 (1974). The nuclearmatrix consists of approximately 10% of the nuclear proteins and isvirtually devoid of lipids, DNA and histones. Fey et al., Crit. Rev.Eukaryotic Gene Expression 1: 127-44 (1991).

[0006] A majority of the known NMPs are common to all cell types andphysiologic states. A number of laboratories have identified NMPs whichmay be unique to certain cell types or states. Mitogenic stimulation andthe induction of differentiation have been demonstrated to alter thecomposition of nuclear matrix proteins and structure. The nuclear matrixcontains a number of associated proteins that have been demonstrated tobe involved in transformation. Berezney first showed that the nuclearmatrix is altered in transformation, examining hepatoma nuclear matrixproteins. Berezney et al., Cancer Res. 39: 3031-39 (1979). Fey andPenman demonstrated that tumor promoters induce a specific morphologicsignature in the nuclear matrix-intermediate filament scaffold of kidneycells. Fey et al., Proc. Nat'l Acad. Sci. USA 81: 859-66 (1984). Fey andPenman went on to demonstrate that the pattern of NMPs differed betweennormal and tumorigenic cell lines. Fey et al., loc. cit. 85: 121-25(1989). An antibody to a nuclear matrix protein, termed NM-200.4, wasraised from the breast carcinoma cell line T-47D. Weidner et al., Am. J.Path. 138: 1293-98 (1991). This antibody reacts strongly with humanbreast carcinoma specimens as well as specimens from lung, thyroid, andovarian cancers, but does not react with normal epithelial cells ofsimilar origin, raising the possibility of the use of certain anti-NMPantibodies as diagnostic tools.

[0007] Exposure of canine kidney cells to various tumor promoters hasalso been found to alter the nuclear matrix-intermediate filamentorganization in these epithelial cells. Fey et al., Proc. Nat'l Acad.Sci. USA 81: 4409-4413 (1984).

[0008] It has been demonstrated with the Dunning rat model of prostatecancer that nuclear matrix protein composition is altered when comparingthe normal dorsal prostate with the spontaneously arisen rat prostateadenocarcinomas. In co-pending U.S. application Ser. No. 08/015,624, theentire contents of which are incorporated by reference herein, whenhuman prostate samples were examined, nuclear matrix proteins wereidentified that (1) were present only in the normal prostate and weremissing in both prostate cancer and benign prostatic hyperplasia (BPH)(normal pattern), (2) were found only in the prostate cancer cells andmissing in the normal prostate and BPH (prostate cancer pattern), and(3) were found in both normal and BPH samples but were absent fromprostate cancers.

[0009] In co-pending U.S. application Ser. No. 08/742,850, the entirecontents of which are incorporated by reference herein, NMPs aredisclosed which are present only in normal bladder cells and are absentin bladder cancer cells. Also, the application discloses other NMPs thatare found only in bladder cancer cells but are absent in normal bladdercells.

[0010] No nuclear matrix proteins have been isolated heretofore,however, that are linked specifically to renal cancer.

SUMMARY OF THE INVENTION

[0011] The present invention relates to nuclear matrix proteins that areable to differentiate cancerous renal cells from normal renal cells,polynucleotide sequences encoding them, and their methods of use. Fiveproteins, respectively designated RCCA-1, RCCA-2, RCCA-3, RCCA-4, andRCCA-5, have been discovered that are present in all cancerous renalcells that are not present in the normal renal cells, and one protein(referred to as RCNL-1) has been discovered that is unique to normalrenal tissue. These proteins are useful for diagnosing and producingtreatments for cell proliferative disorders of the kidney.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0012] According to one aspect, the present invention is directed to apurified nuclear matrix protein or a fragment thereof, which is presentin normal renal cells but absent in cancerous renal cells, or which isabsent in normal renal cells but present in cancerous renal cells. Inparticular, the present invention relates to a protein that is presentin normal renal cells but absent in cancerous renal cells, which isRCNL-1. In addition, the present invention relates to a protein that isabsent in normal renal cells but present in cancerous renal cellsselected from the group consisting of RCCA-1, RCCA-2, RCCA-3, RCCA-4,and RCCA-5.

[0013] Another embodiment of the present invention is a purifiedpolynucleotide sequence encoding the above identified NMPs or NMPfragments of the preceding embodiment. Another embodiment is a purifiedpolynucleotide sequence which hybridizes to the polynucleotide sequenceencoding the above-mentioned NMPs or NMP fragments.

[0014] Another embodiment is a host cell transformed with apolynucleotide sequence encoding the above-mentioned NMPs or NMPfragments. Transformation of a host cell with recombinant DNA may becarried out by conventional techniques known in the art. Where the hostis prokaryotic, such as E. coli, competent cells which are capable ofDNA uptake can be prepared from cells harvested after the exponentialgrowth phase and subsequently treated by the CaCl₂ method by procedureswell known in the art. Alternatively, MgCl₂ or RbCl can be used.Transformation can also be performed after forming a protoplast of thehost cell or by electroporation.

[0015] When the host is a eukaryote, such methods of transfection of DNAas calcium phosphate co-precipitates, conventional mechanical proceduressuch as micro-injection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the NMPs of the invention, anda second foreign DNA molecule encoding a selectable phenotype, such asthe herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein. EUKARYOTIC VIRAL VECTORS, Gluzman (ed.), ColdSpring Harbor Laboratory, 1982.

[0016] Isolation and purification of the NMPs or NMP fragments expressedby a transformed host may be carried out by conventional means includingpreparative chromatography and immunological separations involvingmonoclonal or polyclonal antibodies. Antibodies provided in the presentinvention are immunoreactive with NMP polypeptide or fragments thereof.

[0017] Another embodiment of the invention comprehends a recombinantexpression vector containing the above-mentioned polynucleotidesequences. Preferably, the vector is a virus. Preferred viruses are RNAviruses and preferred RNA viruses are retroviruses. Another preferredvector is a liposome, preferably a target-specific liposome which may betargeted with, for example, an antibody or ligand. Another preferredvector is a plasmid.

[0018] A further embodiment of the invention is an antibody which bindsto the above-mentioned NMPs or NMP fragments. The antibody may bepolyclonal or monoclonal.

[0019] Yet another embodiment is a method for detecting a cellproliferative disorder in a subject, comprising contacting a cellularcomponent from the subject with an antibody or nucleic acid probe whichbinds to a cellular component associated with the cell proliferativedisorder. Preferably, the cellular component is taken from the subject'skidney and is preferably nucleic acid. Preferably, the nucleic acid isDNA encoding the above-mentioned NMPs or NMP fragments. Also preferredas a nucleic acid is RNA. Another preferred cellular component is theabove-mentioned NMPs or NMP fragments.

[0020] Preferably, the nucleic acid probe specifically hybridizes to theabove-mentioned cellular component. When the reagent is a nucleic acidprobe, it is preferably detectably labeled. Preferred labels include aradioisotope, a bioluminescent compound, a chemiluminescent compound, afluorescent compound, a metal chelate, and an enzyme.

[0021] Alternatively, if the cellular component is an NMP or NMPfragment, then an antibody is used which specifically binds to the NMPor NMP fragment. As noted above, the antibody may be monoclonal orpolyclonal.

[0022] Another embodiment is a method of treating a cell proliferativedisorder associated with a protein selected from the group consisting ofRCCA-1, RCCA-2, RCCA-3, RCCA-4, RCCA-5, and RCNL-1, comprisingadministering to a subject with the disorder a therapeutically effectiveamount of an antisense polynucleotide sequence that blocks the sequencesencoding the above-mentioned NMPs. In this embodiment, the treatment isdesigned to block the expression of one or more NMPs which give rise tothe cell proliferative disorder.

[0023] In an alternative method of treatment, instead of using anantisense polynucleotide sequence, a polynucleotide sequence is usedwhich encodes one of the above-mentioned NMPs. In this embodiment, thetreatment is designed to provide the subject with one or more NMPs thatprevent or ameliorate the cell proliferative disorder.

[0024] In another method of treatment, an antibody is administered tothe subject which is capable of blocking the function of one or more ofthe above NMPs.

[0025] Another embodiment is a method of gene therapy, comprisingintroducing into the cells of a host subject an expression vectorcomprising a polynucleotide sequence encoding one or more of theabove-mentioned NMPs. Preferably, the expression vector is introducedinto the cells of the host subject ex vivo, yielding transformed cells,and the transformed cells then are reintroduced into the subject. Apreferred expression vector for this purpose is an RNA virus, preferablya retrovirus.

[0026] The present invention also relates to a method for identifying acomposition which blocks or enhances the function of a renal cell NMP.This embodiment comprises:

[0027] (a) incubating NMP-containing renal cells with a test compositionunder conditions that allow the renal cells and test composition tointeract, and then

[0028] (b) measuring whether the test composition blocks or enhances thefunction of the renal cell NMP.

[0029] Still another embodiment is a kit for detecting acell-proliferative disorder of the kidney comprising a nucleic acidprobe that binds to a polynucleotide sequence encoding one of theabove-mentioned NMPs. Preferably, the probe is labeled for ease ofdetection with a label as described above. Alternatively, the kit maycomprise an antibody which specifically binds to one of theabove-mentioned NMPs. Still another alternative is to use anoligonucleotide primer in the kit that permits amplification of a targetpolynucleotide sequence encoding one of the above-mentioned NMPs, forexample, by polymerase chain reaction (PCR) amplification. Preferably,the kit further includes printed instructions for using the probe orantibody and/or other reagents contained in the kit. Suitable carrierscan be provided in the kits which maintain the probes and/or antibodiesin active form prior to use.

[0030] The NMPs of the present invention include fragments andconservatively substituted variants thereof. Minor modifications of theNMP primary amino acid sequence may result in proteins which havesubstantially equivalent activity as compared to the NMP polypeptidedescribed herein. Such modifications may be deliberate, as bysite-directed mutagenesis, or may be spontaneous. Such modificationsinclude deletion of non-essential amino acids. All of the polypeptidesproduced by these modifications are included herein as long as thebiological activity of the native NMP still exists. Further, deletion ofone or more amino acids can also result in a modification of thestructure of the resultant molecule without significantly altering itsbiological activity. This can lead to the development of a smalleractive molecule which would have broader utility.

[0031] The term “conservative substitution” as used herein denotes thereplacement of an amino acid residue by a structurally similar residue.Examples of conservative substitutions include the substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic for aspartic acids,or glutamine for asparagine, and the like.

[0032] Peptides of the invention can be synthesized by the well knownsolid phase peptide synthesis methods described, for example, byMerrifield, J. Am. Chem. Soc. 85: 2149 (1962), and by Stewart and Young,SOLID PHASE PEPTIDES SYNTHESIS 27-62 (Freeman Publ., 1969).

[0033] The polyclonal and monoclonal antibodies of the invention areimmunoreactive with the NMPs or immunogenic fragments of the NMPs. Ifdesired, polyclonal antibodies can be further purified, for example, bybinding to and elution from a matrix to which NMP polypeptide is boundor by utilizing common nuclear matrix proteins to selectively removenon-specific antibodies. Antibody which consists essentially of pooledmonoclonal antibodies with different epitopic specificities, as well asdistinct monoclonal antibody preparations are provided. The term“antibody” as used in this invention includes intact molecules as wellas fragments thereof, such as Fab and F(ab′)₂ fragments, which arefunctionally capable of binding an epitopic determinant of an NMP.

[0034] A preferred method for the identification and isolation ofantibody binding domains which exhibit binding with NMP is thebacteriophage λ vector system. This vector system has been used toexpress a combinatorial library of Fab fragments from the mouse antibodyrepertoire in Escherichia coli, see Huse et al., Science 246: 1275-81(1989), and from the human antibody repertoire. Mullinax et al., Proc.Nat'l Acad. Sci. USA 87: 8095-99 (1990).

[0035] The phrase “cell-proliferative disorder” here denotes malignantas well as non-malignant (or benign) disorders of the kidney. Thisphrase further encompasses hyperplastic disorders of the kidney. Thecells comprising these proliferative disorders often appearmorphologically and genotypically to differ from the surrounding normaltissue. As noted above, cell-proliferative disorders may be associated,for example, with expression or absence of expression of the NMPs of theinvention. Expression of an NMP at an inappropriate time during the cellcycle or in an incorrect cell type may result in a cell-proliferativedisorder. The NMP-encoding polynucleotide in the form of an antisensepolynucleotide is useful in treating hyperplasia and malignancies of thekidney. When the cell-proliferative disorder is associated with NMPexpression, (e.g., RCCA-1, 2, 3, 4 and/or 5), an antisense NMPpolynucleotide sequence or NMP binding antibody can be introduced intothe renal cells to block the expression and/or function of the NMP.Alternatively, when the cell proliferative disorder is associated withunder-expression or expression of a mutant NMP polypeptide (e.g., RCNL1), a polynucleotide sequence encoding the missing or under-expressedNMP can be introduced into the cell.

[0036] For purposes of the invention, an antibody or nucleic acid probespecific for an NMP may be used to detect the presence of the NMPpolypeptide (in the case of an antibody probe) or polynucleotide (in thecase of the nucleic acid probe) in biological fluids or tissuessuspected of containing the NMP. Oligonucleotide primers based on anycoding sequence region in the NMP sequence are useful for amplifying DNAor RNA, for example by PCR. Any specimen containing a detectable amountof antigen can be used. A preferred sample of this invention is tissuetaken from the kidney. Alternatively, biological fluids which maycontain cells of the kidney may be used.

[0037] The term “subject” as used in this description to denote mammals,preferably humans.

[0038] Another technique which may also result in greater sensitivityconsists of coupling the probe to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactswith avidin, or dinitrophenol, pyridoxal, and fluorescein, which canreact with specific antihapten antibodies.

[0039] The method for detecting a cell expressing a particular NMP ofthe invention or a cell-proliferative disorder associated with an NMP,described above, can be utilized for detection of residual kidney canceror other malignancies or benign hyperplasia conditions in a subject in astate of clinical remission. Additionally, the method for detecting anNMP polypeptide in cells is useful for detecting a cell-proliferativedisorder by identifying cells expressing specific NMPs in comparisonwith NMPs expressed in normal cells. Using the method of the invention,NMP expression can be identified in a cell and the appropriate course oftreatment can be employed (e.g., NMP-encoding or antisense gene therapy,as well as conventional chemotherapy). Since the expression pattern ofthe NMPs of the invention vary with the stage of malignancy of a cell, asample of renal tissue can be screened with a panel of NMP-specificreagents (e.g., nucleic acid probes or antibodies to NMPs) to detect NMPexpression and diagnose the stage of malignancy of the cell.

[0040] The monoclonal antibodies of the present invention are suitablefor use in immunoassays in which they can be utilized in liquid phase orbound to a solid-phase carrier. In addition, the monoclonal antibodiesin these immunoassays can be detectably labeled in various ways.Examples of types of immunoassays which can utilize monoclonalantibodies of the invention are competitive and non-competitiveimmunoassays in either a direct or indirect format. Examples of suchimmunoassays are the radioimmunoassay (RIA) and the sandwich(immunometric) assay. Detection of the antigens using the monoclonalantibodies of the invention can be performed utilizing immunoassayswhich are run in either the forward, reverse, or simultaneous modes,including immunohistochemical assays on physiological samples.Alternatively, the antibody of the invention can be used to detect NMPspresent in electrophoretically dispersed gel protocols such as Westernblots and two-dimensional gels.

[0041] The monoclonal antibodies of the invention can be bound to manydifferent carriers and used to detect the presence of NMP. Examples ofwell-known carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses and magnetite. The nature of the carrier canbe either soluble or insoluble for purposes of the invention.

[0042] In performing the assays it may be desirable to include certain“blockers” in the incubation medium (usually added with the labeledsoluble antibody). The “blockers” are added to assure that non-specificproteins, proteases, or anti-heterophilic immunoglobulins to anti-NMPimmunoglobulins present in the experimental sample do not cross-link ordestroy the antibodies on the solid phase support, or the radiolabeledindicator antibody, to yield false positive or false negative results.The selection of “blockers” therefore may add substantially to thespecificity of the assays described in the present invention.

[0043] It has been found that a number of nonrelevant (i.e.,nonspecific) antibodies of the same class or subclass (isotype) as thoseused in the assays (e.g., IgG1, IgG2a, IgM, etc.) can be used as“blockers.” The “blockers” are used at a level high enough to maintainthe proper sensitivity yet inhibit any unwanted interference by mutuallyoccurring cross reactive proteins in the specimen (normally 1-100μg/μl).

[0044] In this description, the term “epitope” denotes any determinantcapable of specific interaction with the monoclonal antibodies of theinvention. Epitopic determinants usually comprise chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics.

[0045] In using the monoclonal antibodies of the invention for the invivo detection of antigen, the detectably labeled monoclonal antibody isgiven in a dose which is diagnostically effective. The term“diagnostically effective” means that the amount of delectably labeledmonoclonal antibody is administered in sufficient quantity to enabledetection of the site having the NMP antigen for which the monoclonalantibody is specific. The dosage of detectably labeled monoclonalantibody for in vivo diagnosis will vary depending on such factors asage, sex, and extent of disease of the individual. The dosage ofmonoclonal antibody can vary from about 0.001 mg/m², to about 500 mg/m²,preferably 0.1 mg/m² to about 200 mg/m², most preferably about 0.1 mg/m²to about 10 mg/m². Such dosages may vary, for example, depending onwhether multiple injections are given, tumor burden, and other factors.

[0046] For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that the half-life of theradioisotope be long enough so that it is still detectable at the timeof maximum uptake by the target, but short enough so that deleteriousradiation with respect to the host is minimized. Ideally, a radioisotopeused for in vivo imaging will lack a particle emission, but produce alarge number of photons in the 140-250 keV range, which may be readilydetected by conventional gamma cameras.

[0047] For in vivo diagnosis, radioisotopes may be bound toimmunoglobulin either directly or indirectly by using an intermediatefunctional group. Intermediate functional groups which often are used tobind radioisotopes which exist as metallic ions to immunoglobulins arethe bifunctional chelating agents such as diethylenetriamine-pentaceticacid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similarmolecules. Typical examples of metallic ions which can be bound to themonoclonal antibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga,⁷²As, ⁸⁹Zr, and ²⁰¹Tl.

[0048] The monoclonal antibodies of the invention can also be labeledwith a paramagnetic isotope for purposes of in vivo diagnosis, as inmagnetic resonance imaging (MRI) or electron spin resonance (ESR). Ingeneral, any conventional method for visualizing diagnostic imaging canbe utilized. Usually gamma and positron emitting radioisotopes are usedfor camera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr,and ⁵⁶Fe.

[0049] The monoclonal antibodies of the invention can be used to monitorthe course of amelioration of an NMP-associated cell-proliferativedisorder. Thus, by measuring the increase or decrease in the number ofcells expressing a NMP or changes in NMP present in various body fluids,such as ejaculate or urine, it would be possible to determine whether aparticular therapeutic regimen aimed at ameliorating the disorder iseffective.

[0050] The monoclonal antibodies of the invention can also be used,alone or in combination with effector cells, see Douillard et al.,Hybridoma 5 (Supp. 1): S139 (1986), for immunotherapy in an animalhaving a cell proliferative disorder which expresses NMP polypeptidewith epitopes reactive with the monoclonal antibodies of the invention.

[0051] When used for immunotherapy, the monoclonal antibodies of theinvention may be unlabeled or attached to a therapeutic agent. Theseagents can be coupled either directly or indirectly to the monoclonalantibodies of the invention. One example of indirect coupling is by useof a spacer moiety. These spacer moieties, in turn, can be eitherinsoluble or soluble, see Diener et al., Science 231: 148 (1986), andcan be selected to enable drug release from the monoclonal antibodymolecule at the target site. Examples of therapeutic agents which can becoupled to the monoclonal antibodies of the invention for immunotherapyare drugs, radioisotopes, lectins, and toxins.

[0052] Non-proteinaceous as well as proteinaceous drugs can beconjugated to the monoclonal antibodies of the present invention.Preferred drugs for conjugation include mitomycin C, daunorubicin,vinblastine, and others used to treat cancer.

[0053] The proteinaceous drugs with which the monoclonal antibodies ofthe invention can be joined include immunomodulators and otherbiological response modifiers. The term “biological response modifiers”encompasses substances which are involved in modifying the immuneresponse in such manner as to enhance the destruction of anNMP-associated tumor for which the monoclonal antibodies of theinvention are specific. Examples of immune response modifiers includesuch compounds as lymphokines. Lymphokines include tumor necrosisfactor, the interleukins, lymphotoxin, macrophage activating factor,migration inhibition factor, colony stimulating factor, and interferon.Interferons with which the monoclonal antibodies of the invention can belabeled include alpha-interferon, beta-interferon and gamma-interferonand their subtypes.

[0054] In using radioisotopically conjugated monoclonal antibodies ofthe invention for immunotherapy certain isotopes may be more preferablethan others depending on such factors as tumor cell distribution as wellas isotope stability and emission. If desired, the tumor celldistribution can be evaluated by the in vivo diagnostic techniquesdescribed above. Depending on the cell proliferative disease someemitters may be preferable to others. In general, alpha and betaparticle-emitting radioisotopes are preferred in immunotherapy. Forexample, if an animal has solid tumor foci a high energy beta emittercapable of penetrating several millimeters of tissue, such as ⁹⁰Y may bepreferable. On the other hand, if the cell proliferative disorderconsists of simple target cells, as in the case of leukemia, a shortrange, high energy alpha emitter, such as ²¹²Bi, may be preferable.Examples of radioisotopes which can be bound to the monoclonalantibodies of the invention for therapeutic purposes are 125I, 131I ⁹⁰Y,⁶⁷Cu, ²¹²Bi, ²¹¹At, ²¹²pb, ⁴⁷SC, ¹⁰⁹Pd, ⁶⁵Zn, and Re.

[0055] Lectins are proteins, usually isolated from plant material, whichbind to specific sugar moieties. Many lectins are also able toagglutinate cells and stimulate lymphocytes. Ricin is a toxic lectinwhich has been used immunotherapeutically. The alpha-peptide chain ofricin, which is responsible for toxicity, may be bound to the antibodyof the invention to enable site specific delivery of the toxic effect.

[0056] Toxins are poisonous substances produced by plants, animals, ormicroorganisms, that, in sufficient dose, are often lethal. Diphtheriatoxin is a substance produced by Corynebacterium diphtheria which can beused therapeutically. This toxin consists of an alpha and beta subunitwhich under proper conditions can be separated. The toxic A componentcan be bound to an antibody and used for site specific delivery to a NMPbearing cell.

[0057] The monoclonal antibodies of the invention can be used incombination with alpha-interferon. This treatment modality enhancesmonoclonal antibody targeting of carcinomas by increasing the expressionof monoclonal antibody reactive antigen by the carcinoma cells. Greineret al., Science 235: 895 (1987). Alternatively, the monoclonal antibodyof the invention can be used, for example, in combination withgamma-interferon to thereby activate and increase the expression of Fcreceptors by effector cells which, in turn, results in an enhancedbinding of the monoclonal antibody to the effector cell and killing oftarget tumor cells.

[0058] It is also possible to utilize liposomes with the monoclonalantibodies of the invention in their membrane to specifically deliverthe liposome to the tumor expressing NMP. These liposomes can beproduced such that they contain, in addition to the monoclonal antibody,such immunotherapeutic agents as those described above which would thenbe released at the tumor site. Wolff et al., Biochem. Biophys. Acta 802:259 (1984).

[0059] The dosage ranges for the administration of monoclonal antibodiesof the invention are those large enough to produce the desired effect inwhich the symptoms of the malignant disease are ameliorated. The dosageshould not be so large as to cause adverse side effects, such asunwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the patient and can be determined by one of skill inthe art. The dosage can be adjusted by the individual physician in theevent of any complication. Dosage can vary from about 0.1 mg/kg to about2000 mg/kg, preferably about 0.1 mg/kg to about 500 mg/kg, in one ormore dose administrations daily, for one or several days. Generally,when the monoclonal antibodies of the invention are administeredconjugated with therapeutic agents, lower dosages, comparable to thoseused for in vivo diagnostic imaging, can be used.

[0060] The monoclonal antibodies of the invention can be administeredparenterally by injection or by gradual perfusion over time. Themonoclonal antibodies of the invention can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intracavity, or transdermally, alone or in combination with effectorcells.

[0061] The present invention also provides a method for treating asubject with an NMP-associated cell-proliferative disorder using an NMPnucleotide sequence. An NMP nucleotide sequence which may encode asuppressor polypeptide may be under-expressed as compared to expressionin a normal cell, therefore it is possible to design appropriatetherapeutic or diagnostic techniques directed to this sequence. Thus,where a cell-proliferative disorder is associated with the expression ofan NMP associated with malignancy, nucleic acid sequences that interferewith NMP expression at the translational level can be used. Thisapproach utilizes, for example, antisense nucleic acid and ribozymes toblock translation of a specific NMP mRNA, either by masking that mRNAwith an antisense nucleic acid or by cleaving it with a ribozyme. Incases when a cell proliferative disorder or abnormal cell phenotype isassociated with the under expression of NMP suppressor for example,nucleic acid sequences encoding NMP (sense) could be administered to thesubject with the disorder.

[0062] Antisense nucleic acids are DNA or RNA molecules that arecomplementary to at least a portion of a specific MRNA molecule.Weintaub, Scientific American, 262: 40 (1990). In the cell, theantisense nucleic acids hybridize to the corresponding mRNA, forming adouble-stranded molecule. The antisense nucleic acids interfere with thetranslation of the mRNA since the cell will not translate a mRNA that isdouble-stranded. Antisense oligomers of about 15 nucleotides arepreferred, since they are easily synthesized and are less likely to beexpressed than larger molecules when introduced into the targetNMP-producing cell.

[0063] Ribozymes are RNA molecules possessing the ability tospecifically cleave other single-stranded RNA in a manner analogous toDNA restriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it. Cech, J. Amer. Med. Assn. 260: 3030 (1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

[0064] There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

[0065] The present invention also provides gene therapy for thetreatment of cell proliferative disorders which are mediated by NMP.Such therapy requires introduction of the appropriate NMP polynucleotidesequence (antisense or encoding strand) into cells of subjects havingthe proliferative disorder. Delivery of antisense NMP polynucleotidescan be achieved using a recombinant expression vector such as a chimericvirus or a liposome. Disorders associated with under-expression of anNMP or expression of a cancer-associated NMP can be treated using genetherapy with the encoding or antisense nucleotide sequences,respectively.

[0066] Various viral vectors which can be utilized for gene therapy astaught herein include adenovirus, herpes virus, vaccinia, or,preferably, an RNA virus such as a retrovirus. Preferably, theretroviral vector is a derivative of a murine or avian retrovirus.Examples of retroviral vectors in which a single foreign gene can beinserted include, but are not limited to: Moloney murine leukemia virus(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additionalretroviral vectors can incorporate multiple genes. All of these vectorscan transfer or incorporate a gene for a selectable marker so thattransduced cells can be identified and generated. By inserting an NMPsequence of interest into the viral vector along with another gene whichencodes the ligand for a receptor on a specific target cell, forexample, the vector is rendered target specific. Retroviral vectors canbe made target specific by inserting, for example, a polynucleotideencoding a sugar, a glycolipid, or a protein. Preferred targeting isaccomplished by using an antibody to target the retroviral vector.

[0067] Since recombinant retroviruses are defective in one or moregenes, they require assistance in order to produce infectious vectorparticles. Helper cell lines which have deletions of the packagingsignal include but are not limited to Ψ2, PA317 and PA12, for example.These cell lines produce empty virions, since no genome is packaged. Ifa retroviral vector is introduced into such cells in which the packagingsignal is intact, but the structural genes are replaced by other genesof interest, the vector can be packaged and vector virion produced.

[0068] Alternatively, NIH 3T3 or other tissue culture cells can bedirectly transfected with plasmids encoding the retroviral structuralgenes gag, pol and env by conventional calcium phosphate transfection.These cells are then transfected with the vector plasmid containing thegenes of interest. The resulting cells release the retroviral vectorinto the culture medium.

[0069] Other targeted delivery systems for NMP antisense polynucleotidesinclude macromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. It has beenshown that large unilamellar vesicles (ULV), which range in size from0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffercontaining large macromolecules. RNA, DNA and intact virions can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form. Fraley et al., Trends Biochem. Sci. 6: 77(1981).

[0070] The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

[0071] Examples of lipids useful in liposome production includephosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phosphatidyiserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

[0072] The targeting of liposomes has been classified based onanatomical and mechanistic factors. Anatomical classification is basedon the level of selectivity, for example, organ-specific, cell-specificand organelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

[0073] The surface of the targeted delivery system may be modified in avariety of ways. In the case of a hyposomal targeted delivery system,lipid groups can be incorporated into the lipid bilayer of the liposomein order to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

[0074] In general, the compounds bound to the surface of the targeteddelivery system will be ligands and receptors which will allow thetargeted delivery system to find and “home in” on the desired cells. Aligand may be any compound of interest which will bind to anothercompound, such as a receptor.

[0075] In general, surface membrane proteins which bind to specificeffector molecules are referred to as receptors. In the presentinvention, antibodies of the invention are preferred receptors.Antibodies can be used to target liposomes to specific cell-surfaceligands, in this case the NMPs of choice. Preferably, the target tissueis renal tissue. A number of procedures can be used to covalently attacheither polyclonal or monoclonal antibodies to a liposome bilayer.Antibody-targeted liposomes can include monoclonal or polygonalantibodies or fragments thereof such as Fab, or F(ab′)₂, as long as theybind efficiently to an the antigenic epitope on the target cells.

[0076] Preparations for parenteral administration include sterileaqueous or nonaqueous solutions, suspensions, and emulsions. Examples ofnon aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such an those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, antimicrobials, anti-oxidants, chelating agents and inert gasesand the like.

[0077] The invention also relates to a method for preparing a medicamentor pharmaceutical composition comprising the polynucleotides or themonoclonal antibodies of the invention, the medicament being used fortherapy of NMP associated cell proliferative disorders.

[0078] The invention is further illustrated by the following,non-limiting examples.

[0079] Tissue Samples and Cell Lines

[0080] Human tissue samples were obtained from patients undergoingradical nephrectomy for previously diagnosed renal cell cancer at theUniversity of Pittsburgh Medical Center. Matched tumor and normal kidneytissue was obtained from 17 patients. Samples were taken afterdetermination of tumor and normal kidney by gross examination andconfirmed by histologic examination of tissue adjacent to the sampledareas. The tissue samples were placed in PBS with 1 mMphenylmethylsulfonyl fluoride (PMSF) and stored at −70° C. until suchtime that they were processed to extract the nuclear matrix proteins.Tumors were staged according to the TNM system and nuclear grades wereassigned according to the Fuhrman system. Tumor stages and grades of the17 tumors are shown in Table 1. TABLE 1 Pathologic stage and grade ofthe tumors studied Patient No. Tumor Type TNM stage and grade 1 Clearcell and papillary T2NxMx; II/IV 2 Clear cell T3bN1Mx; III/IV 3 Clearcell T2NoMx; I/IV 4 Granular T3bNoMx; II/IV 5 Clear cell and granularT1NxMx; II/IV 6 Clear cell T3aNxMx; II/IV 7 Clear cell and sarcomatoidT3bNxMx; II and III/IV 8 Clear T2NoMx; II/IV 9 Granular and clear cellT2NxMx; II/IV 10 Clear cell T2NxMx; I/IV 11 Clear cell T3bNxMx; II/IV 12Clear cell (areas of T2NoMx; II/Iv chromophobe adenoma) 13 Clear cellT2NxMx; II/IV 14 Poorly differentiated T4N2M1; IV/IV 15 Clear cell andgranular T2NxMx; II/IV 16 Clear cell T4N1M1; III/IV 17 Clear cellT3NxMx; II/IV

[0081] Two cell lines A498 and 769-P obtained from ATCC (Rockville, Md.)were also used for this study. Both cell lines had been established fromprimary renal cell carcinomas, 769-P from the clear cell type and A498from papillary RCC. The 769-P cells were grown in RPMI ₁₆₄₀TM medium(GIBCO BRL, Life Technologies, Grand Island, N.Y.) supplemented withL-glutamine, 10% fetal bovine serum, HEPES, sodium pyruvate and 1%penicillin-streptomycin. The A-498 cells were cultivated in modifiedEAGLES™ medium (GIBCO BRL, Life Technologies, Grand Island, N.Y.) withEarle's basic salt solution and non-essential amino acids supplementedwith 10% fetal bovine serum, sodium pyruvate and 1%penicillin-streptomycin.

[0082] Nuclear Matrix Preparation

[0083] The nuclear matrix proteins (NMPs) were extracted from thetissues and cell lines according to the following method. First,cytoskeletal and nuclear membrane components were removed by saltextraction and treatment with ammonium sulfate. The nuclear chromatinwas then removed by using DNase I and RNase, leaving behind the nuclearmatrix components.

[0084] The pieces of tissue were minced into small (<1 mm³) pieces andhomogenized with a Teflon pestle on ice with 0.5% Triton X-100 in asolution containing 2 mM vanadyl ribonucleoside (Rnase inhibitor) torelease the lipids and soluble proteins. The homogenized tissue was thenfiltered through a 350 μm nylon mesh and treated with 0.25M ammoniumsulfate to remove the soluble cytoskeletal components. Dnase and Rnasetreatment was then used to remove the chromatin. The remaining fractioncontained intermediate filaments and NMPs. This was then disassembledwith 8M urea and the insoluble components consisting of carbohydratesand extracellular matrix were pelleted out. After dialyzing the ureaout, the intermediate filaments were allowed to reassemble and weresubsequently removed by centrifugation. The NMPs were precipitated inethanol. Freshly prepared PMSF was added to all solutions to preventdigestion by serine proteases, 0.3 μM aprotonin, 1 μM leupeptin and 1 μMpepstatin. The protein concentration was determined by resuspending theproteins in PBS or sample buffer and using the Coomassie Plus proteinassay using bovine serum albumin as a standard. For electrophoresis, theNMPs which were precipitated in ethanol were dissolved in a samplebuffer consisting of 9M urea, 65 mM3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate, 2.2%ampholytes and 140 mM DTT (ESA Inc., Chelmsford, Mass.). The finalpellet containing the NMPs represented <1% of the total cellularproteins.

[0085] High Resolution Two-Dimensional Electrophoresis

[0086] Electrophoresis was performed using the INVESTIGATOR 2-D^(TM) gelsystem (ESA Inc., Chelmsford, Mass.). One-dimensional isoelectricfocusing was carried out for 18,000 volt-hours using 1 mm×18 cm tubegels after 1.5 hours of prefocusing. The tube gels were extruded andplaced on top of 1 mm SDS DURACRYL™ (ESA Inc., Chelmsford, Mass.) hightensile strength PAGE slab gels. The gels were electrophoresed at 12° C.constant temperature for 4.5 to 5 hours. Gels were fixed with 50%methanol and 10% acetic acid. After thorough rinsing and rehydration,gels were treated with 5% glutaraldehyde and 5 mM DTT after bufferingwith 50 mM phosphate (pH 7.2). The gels were stained with silver stain(Accurate Chemical Co., Westbury, N.Y.). Fifty μg of protein were loadedper gel. Protein molecular weight standards were silver standards fromDiversified Technology (Newton Center, Mass.). Isoelectric points weredetermined using carbamylated standards (BDH-distributed byGallard-Schlesinger, Carle Place, N.Y. and Sigma Chemical Co., St.Louis, Mo.). Multiple gels were run for each sample and multiple sampleswere run at different times. Only protein spots clearly and reproduciblyobserved in all the gels of a sample type were taken into account asthose representing NMPs. The gels were analyzed using the 2DELECTROPHORESIS ANALYSIS SYSTEM™ (BioImage, Ann Arbor, Mich.) whichmatches protein spots between gels and sorts the gels and protein spotsinto a database.

[0087] Consistent differences in NMP composition were noted betweennormal kidney tissue and renal cancer cells in all the samples. Fivecharacteristic and unique NMPs were detected by two-dimensionalelectrophoresis in all seventeen tumor samples which were absent in thesamples of normal kidney tissue (RCCA 1-5). These NMPs were found in allthe tumors irrespective of histologic subtype or nuclear grade. In orderto limit the possibility that the differences in NMP composition notedin these studies were due to differences in mitotic rates, tumors ofvarious nuclear grades were selected, including nuclear grade II tumorswhich do not have increased number of mitoses over normal controltissue. One NMP was detected exclusively in all the normal kidneysamples and was absent in all the tumor tissues (RCNL-1). All theproteins isolated appear to be unique and their PIs and molecularweights are completely different from those proteins detected in earlierstudies in prostate, breast and bladder cancers.

[0088] In order to rule out the possibility that the differences in NMPcomposition may be due to the detection of NMPs from stromal and othercell types admixed with the homogenized sample, the NMP composition oftwo renal cancer cell lines was also examined. All five of the NMPsidentified in the human tumor samples (RCCA 1-5) were also found in boththe cell lines.

[0089] The following NMPs were identified: TABLE 2 Molecular Weight (kD)pI Proteins Associated With Human Renal Cancer RCCA-1 53,000 9.30 RCCA-232,000 6.95 RCCA-3 27,000 6.50 RCCA-4 20,000 5.25 RCCA-5 15,000 6.00Protein Associated with Normal Human Kidney RCNL-1 103,000  8.30

[0090] While the invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to oneskilled in the art that various changes and modifications can be madetherein without departing from the spirit and scope of the invention.

What is claimed is:
 1. A purified nuclear matrix protein that is presentin normal renal cells but absent in cancerous renal cells, or that isabsent in normal renal cells but present in cancerous renal cells. 2.The protein or fragment as claimed in claim 1 , wherein the protein isRCNL-1.
 3. The protein or fragment of claim 1 , wherein the protein isselected from the group consisting of RCCA-1, RCCA-2, RCCA-3, RCCA-4,and RCCA-5.
 4. A purified polynucleotide sequence encoding a protein orfragment of claim 1 .
 5. A purified polynucleotide sequence whichhybridizes to the polynucleotide sequence of claim 4 .
 6. A host celltransformed with the polynucleotide of claim 4 .
 7. A recombinantexpression vector containing the polynucleotide of claim 4 .
 8. Thevector of claim 7 , wherein the vector is a virus.
 9. The vector ofclaim 8 , wherein the virus is an RNA virus.
 10. The vector of claim 9 ,wherein the RNA virus is a retrovirus.
 11. The vector of claim 7 ,wherein the vector is a liposome.
 12. The vector of claim 11 , whereinthe liposome is target-specific.
 13. The vector of claim 12 , whereinthe liposome is targeted with an antibody.
 14. The vector of claim 7 ,wherein the vector is a plasmid.
 15. An antibody which binds to theprotein of claim 1 .
 16. A method for detecting a cell proliferativedisorder in a subject, comprising contacting a cellular component fromthe subject with a reagent which binds to a cellular componentassociated with a cell proliferative disorder.
 17. The method of claim16 , wherein the cellular component is taken from the subject's kidney.18. The method of claim 17 , wherein the cellular component is nucleicacid.
 19. The method of claim 18 , wherein the nucleic acid is DNA. 20.The method of claim 18 , wherein the nucleic acid is RNA.
 21. The methodof claim 17 , wherein the cellular component is protein.
 22. The methodof claim 16 , wherein the reagent is a probe.
 23. The method of claim 22, wherein the probe is nucleic acid.
 24. The method of claim 22 ,wherein the probe is an antibody.
 25. The method of claim 24 , whereinthe antibody is polyclonal.
 26. The method of claim 24 , wherein theantibody is monoclonal.
 27. The method of claim 22 , wherein the probeis detectably labeled.
 28. The method of claim 27 , wherein the label isselected from the group consisting of a radioisotope, a bioluminescentcompound, a chemiluminescent compound, a fluorescent compound, a metalchelate, and an enzyme.
 29. A method of treating a cell proliferativedisorder associated with a protein selected from the group consisting ofRCCA-1, RCCA-2, RCCA-3, RCCA-4, RCCA-5, and RCNL-1, comprisingadministering to a subject with the disorder a therapeutically effectiveamount of reagent which blocks or enhances the function of the protein.30. The method of claim 29 , wherein the reagent is an antisensepolynucleotide sequence.
 31. The method of claim 29 , wherein thereagent is a polynucleotide sequence encoding a protein selected fromthe group consisting of RCCA-1, RCCA-2, RCCA-3, RCCA-4, RCCA-5, andRCNL-1.
 32. The method of claim 29 , wherein the reagent is an antibody.33. The method of claim 32 , wherein the antibody is monoclonal.
 34. Themethod of claim 29 , wherein the cell proliferative disorder is in renaltissue.
 35. A method of gene therapy, comprising introducing into thecells of a host subject an expression vector comprising a nucleotidesequence encoding a protein of claim 1 .
 36. The method of claim 35 ,wherein the expression vector is introduced into the cells of the hostsubject ex vivo, yielding transformed cells, and the transformed cellsthen are reintroduced into the subject.
 37. The method of claim 36 ,wherein the expression vector is an RNA virus.
 38. The method of claim37 , wherein the RNA virus is a retrovirus.
 39. The method of claim 36 ,wherein the subject is human.
 40. A method for identifying a compositionwhich blocks or enhances the function of an NMP of renal cells, whichmethod comprises: (a) incubating NMP-containing renal cells with a testcomposition under conditions that allow the renal cells and testcomposition to interact, and then (b) measuring whether the testcomposition causes blocking or enhancement of the function of an NMP ofthe renal cells.
 41. The method of claim 40 , wherein the NMP isselected from the group consisting of RCCA-1, RCCA-2, RCCA-3, RCCA-4,RCCA-5, and RCNL-1.
 42. The method of claim 40 , wherein the effectbeing measured is blocking of the function of the NMP.
 43. The method ofclaim 40 , wherein the effect being measured is enhancement of thefunction of the NMP.
 44. A kit useful for the detection of acell-proliferative disorder associated with a protein of claim 1 , saidkit comprising a probe for identifying a protein or fragment of claim 1or a polynucleotide sequence encoding a protein or fragment of claim 1 .45. The kit of claim 44 , wherein the probe is an antibody capable ofbinding to the protein or fragment.
 46. The kit of claim 44 , whereinthe probe is a polynucleotide probe capable of hybridizing with thepolynucleotide sequence encoding the protein or fragment.
 47. The kit ofclaim 46 , further comprising a container containing an oligonucleotideprimer for amplification of the polynucleotide sequence encoding theprotein or fragment.